Bottom Line:
Our characterization of Nse5 establishes a previously unidentified relationship between the Smc5/6 complex and factors of the SUMO pathway.Nse5 physically associates with the E2 conjugating enzyme, Ubc9, where contacts are stabilized by non-covalent interactions with SUMO.Overall, given the extensive connection between Nse5 and components of the SUMO pathway, we speculate that one function of the Smc5/6 complex might be as a scaffold center to enable sumoylation events in budding yeast.

BIO018440F1: Genetic interactions and Smc5 sumoylation with the nse5-ts1 mutant allele of the Smc5/6 complex. (A-C) Cell viability was monitored as colony outgrowth from asynchronous cultures after transient exposure to MMS at indicated concentrations for 1 h at 30°C, with values normalized to survival at time point 0. The % survival reported in graph is value after treatment with 0.03% MMS. Data points represent the mean±s.d. at each concentration of MMS from n=3 experiments performed in technical duplicate. *P<0.01 compared to wild-type cells with values from a two-tailed t-test. Drop assays on YPAD at 37°C or 25°C without or with the indicted amounts of MMS were performed on 1:10 serial dilutions of exponentially growing cultures. The following strains were used: wild-type (WT) (JC471), nse5-ts1 (JC1321), smc5-6 (JC1844), nse5-ts1/smc5-6 (JC1843), smc6-9 (JC1358) and nse5-ts1/smc6-9 (JC1359), mms21-11 (JC1981) and nse5-ts1/mms21-11 (JC1319). (D) Drop assays (1:10 serial dilutions) from exponentially growing cultures were performed on YPAD medium at 37°C or 25°C without or with MMS at the indicated concentration for wild-type (JC1157), nse5-ts1 (JC1321), mms21-11 (JC1981) and nse5-ts1/mms21-11 (JC1319). (E,F) Smc5 sumoylation was analyzed in cells without or with 0.3% MMS at 25°C by immunoprecipitation of Myc-Smc5 followed by western blot analysis with anti-Smt3 at a concentration of 1:3000 (a kind gift from Zhao lab). Blots were then stripped and re-probed using anti-Myc antibody (9E10) at a concentration of 1:1000 to confirm equal loading. In a side-by-side comparison, Smc5 sumoylation increases upon MMS treatment and is reduced in nse5-ts1 cells (JC911) and mms21-11 (JC954) compared to wild-type (JC720). (G) As an alternative method to detect sumoylation, proteins were isolated by Ni-NTA affinity purification of His-Smt3 as described previously at 25°C (Wohlschlegel et al., 2004; Psakhye and Jentsch, 2012; Bustard et al., 2012) followed by western blotting with anti-Myc antibodies to visualize sumoylated proteins in cells containing Myc-tagged Smc5 with un-tagged Smt3 wild-type (JC720), or His8-tagged Smt3 in wild-type (JC1157), nse5-ts1 (JC1156) and mms21-11 (JC1155) after treatment with 0.3% MMS.

Mentions:
The Smc5/6 complex is involved in DNA replication and repair and characterizing the individual subcomponents of the complex will augment full understanding of how Smc5/6 works. To begin our characterization of the Nse5 component, we utilized the ts mutant, nse5-ts1, which is lethal at 37°C (Fig. S1A,B) (Bustard et al., 2012; Ben-Aroya et al., 2008). We combined this allele with other mutants in the Smc5/6 complex. Upon treatment with 0.03% MMS for 1 h, there was a loss in viability to ∼7% when nse5-ts1 was combined with mms21-11 compared to 67% and 96% for the nse5-ts1 and mms21-11 single mutants, respectively (Fig. 1A). This synergistic loss of viability was not observed when nse5-ts1 was combined with two other complex mutants, smc5-6 or smc6-9 (Fig. 1B,C), suggesting a potential for overlap in the functionality of Nse5 and Mms21. This sensitivity was also observed when cells were grown on plates containing a low concentration of 0.001% MMS (Fig. 1D). Other genetic interactions were also observed, for example nse5-ts1/nse3-1 double temperature-sensitive mutants showed moderate sensitivity over the single alleles (Fig. S1C) and cells harboring nse5-ts1/nse4-2 double mutants exhibited MMS sensitivity similar to levels observed with nse5-ts1/mms21-11 (Fig. S1C). In contrast to Mms21, however, a defined role for Nse4 within the complex is currently unclear and therefore was not further investigated in this study. As Mms21 is SUMO ligase (Zhao and Blobel, 2005) and Nse5 has potential ties to the SUMO pathway (Hazbun et al., 2003; Bustard et al., 2012), we wanted to investigate if Nse5 mediates the sumoylation of certain target proteins, such as Smc5, after MMS exposure.Fig. 1.

BIO018440F1: Genetic interactions and Smc5 sumoylation with the nse5-ts1 mutant allele of the Smc5/6 complex. (A-C) Cell viability was monitored as colony outgrowth from asynchronous cultures after transient exposure to MMS at indicated concentrations for 1 h at 30°C, with values normalized to survival at time point 0. The % survival reported in graph is value after treatment with 0.03% MMS. Data points represent the mean±s.d. at each concentration of MMS from n=3 experiments performed in technical duplicate. *P<0.01 compared to wild-type cells with values from a two-tailed t-test. Drop assays on YPAD at 37°C or 25°C without or with the indicted amounts of MMS were performed on 1:10 serial dilutions of exponentially growing cultures. The following strains were used: wild-type (WT) (JC471), nse5-ts1 (JC1321), smc5-6 (JC1844), nse5-ts1/smc5-6 (JC1843), smc6-9 (JC1358) and nse5-ts1/smc6-9 (JC1359), mms21-11 (JC1981) and nse5-ts1/mms21-11 (JC1319). (D) Drop assays (1:10 serial dilutions) from exponentially growing cultures were performed on YPAD medium at 37°C or 25°C without or with MMS at the indicated concentration for wild-type (JC1157), nse5-ts1 (JC1321), mms21-11 (JC1981) and nse5-ts1/mms21-11 (JC1319). (E,F) Smc5 sumoylation was analyzed in cells without or with 0.3% MMS at 25°C by immunoprecipitation of Myc-Smc5 followed by western blot analysis with anti-Smt3 at a concentration of 1:3000 (a kind gift from Zhao lab). Blots were then stripped and re-probed using anti-Myc antibody (9E10) at a concentration of 1:1000 to confirm equal loading. In a side-by-side comparison, Smc5 sumoylation increases upon MMS treatment and is reduced in nse5-ts1 cells (JC911) and mms21-11 (JC954) compared to wild-type (JC720). (G) As an alternative method to detect sumoylation, proteins were isolated by Ni-NTA affinity purification of His-Smt3 as described previously at 25°C (Wohlschlegel et al., 2004; Psakhye and Jentsch, 2012; Bustard et al., 2012) followed by western blotting with anti-Myc antibodies to visualize sumoylated proteins in cells containing Myc-tagged Smc5 with un-tagged Smt3 wild-type (JC720), or His8-tagged Smt3 in wild-type (JC1157), nse5-ts1 (JC1156) and mms21-11 (JC1155) after treatment with 0.3% MMS.

Mentions:
The Smc5/6 complex is involved in DNA replication and repair and characterizing the individual subcomponents of the complex will augment full understanding of how Smc5/6 works. To begin our characterization of the Nse5 component, we utilized the ts mutant, nse5-ts1, which is lethal at 37°C (Fig. S1A,B) (Bustard et al., 2012; Ben-Aroya et al., 2008). We combined this allele with other mutants in the Smc5/6 complex. Upon treatment with 0.03% MMS for 1 h, there was a loss in viability to ∼7% when nse5-ts1 was combined with mms21-11 compared to 67% and 96% for the nse5-ts1 and mms21-11 single mutants, respectively (Fig. 1A). This synergistic loss of viability was not observed when nse5-ts1 was combined with two other complex mutants, smc5-6 or smc6-9 (Fig. 1B,C), suggesting a potential for overlap in the functionality of Nse5 and Mms21. This sensitivity was also observed when cells were grown on plates containing a low concentration of 0.001% MMS (Fig. 1D). Other genetic interactions were also observed, for example nse5-ts1/nse3-1 double temperature-sensitive mutants showed moderate sensitivity over the single alleles (Fig. S1C) and cells harboring nse5-ts1/nse4-2 double mutants exhibited MMS sensitivity similar to levels observed with nse5-ts1/mms21-11 (Fig. S1C). In contrast to Mms21, however, a defined role for Nse4 within the complex is currently unclear and therefore was not further investigated in this study. As Mms21 is SUMO ligase (Zhao and Blobel, 2005) and Nse5 has potential ties to the SUMO pathway (Hazbun et al., 2003; Bustard et al., 2012), we wanted to investigate if Nse5 mediates the sumoylation of certain target proteins, such as Smc5, after MMS exposure.Fig. 1.

Bottom Line:
Our characterization of Nse5 establishes a previously unidentified relationship between the Smc5/6 complex and factors of the SUMO pathway.Nse5 physically associates with the E2 conjugating enzyme, Ubc9, where contacts are stabilized by non-covalent interactions with SUMO.Overall, given the extensive connection between Nse5 and components of the SUMO pathway, we speculate that one function of the Smc5/6 complex might be as a scaffold center to enable sumoylation events in budding yeast.